U.K. Linderstrom-Lang and J.A. Schellman concluded in 1959 that the stability of secondary structure of a protein would depend on the presence of closed loop of primary (or strong non-covalent) bonds and that the location where this loop would be broken would make very little difference. This Lang-Schellman model represents the first prediction of existence of many-body forces stabilizing proteins (e.g. a two-body force is the interaction between two atoms. In contrast, the many-body force is due to the concerted interactions among a set of positioned atoms or atomic groups e.g. the closed loop). However, the many-body force with proteins has not been pursued prehaps because the nature of the atom-atom interactions manifesting themselves in the many-body force (the closed loop) is unclear and it is not easy to map or identify the residues participating in generation of the many-body force (the closed loop). Neverthelass, I believe that the Lang-Schellman many-body force is crucial for the structure-function of proteins and therefore, needs investigation. In previous studies of staphylococcal nuclease, we defined the permissible region for cleavage of a protein. That is, a protein fragment complex with native like structure and function can form only if cleavage of the protein occurs in one of the permissible regions for cleavage, the number of which is limited. The existence of only a limited number of such permissible regions for cleavage of a protein is consistent with the characteristics of the Lang-Schellman many-body force and therefore, supports the presence of the Lang-Schellman many-body force. Thus, we have been studying the nature of the interatomic interactions that manifest themselves in the Lang-Schellman many-body force. For this, the cytochrome c (cyt.c) system has been an excellent test object. Others have shown that the unfolding free energy is greater by 5.2 and 4.2 kcal per mol, respectively for horse cyt. c than for yeast iso-1- and iso-2- cyts. c and that the origin of this difference is entropic. Our previous studies show that the difference in hydrophobicity of the hydrophobic core residues dose not account for a majority of the stability difference between horse cyt. c and iso-2 and suggest that instead, interactions between the hydrophobic core residues and surface residues, operative after formation of a majority of the hydrophobic core, may be important for such a stability difference. As described in the previous year, we have obtained evidence that the hydrophobic core of cyt.c is sensitive to the surface charge. A combination of these our observations and the Tsao-Evans-Wennerstrom polarizable domain model for interactions between two ordered hydrophobic monolayers has led us to the following model as reported in the previous year. There is a domain associated with the ordered hydrophobic core of cyt. c that would be polarizable. This domain would respond through its polarization to the electric field of the surface charges in such a way that the protein would be stabilized. Thus, our polarizable hydrophobic core domain model provides for the first time an explanation for the Lang-Schellman many-body force. Recent observations by others with reduced Rnase A and the three-disulfide bonded Rnase A, if combined with our previous observations and those of others with Rnase A and its derivatives, support our polarizable hydrophobic core domain model. To prove our model, it is necessary to map or identify the residues constituting the polarizable hydrophobic core domain, which would respond to the electric field of the surface charges, and those residues representing such surface charges. We assume that the stability difference between horse and iso-2 cyts. c is essentially due to the difference in such core-surface electrostatic interactions. Therefore, the task is to identify those surface and core residues, that are involved in such core-surface electrostatic interactions stabilizing more horse cyt. c than iso-2. To begin this mapping, we are preparing an iso-2 cyt c mutant in which all hydrophobic core residues and most of the ionizable surface residues correspond to those corresponding of horse cyt. c. This is to see whether the stability of such a mutant is close to horse cyt. c rather than iso-2. To this end, of the 25 fold (times) planned amino acid residue mutations of iso-2, 23 times are now achieved in terms of mutation of the iso-2 gene contained in the phagemid. It is planned that expression of a set of such mutant genes would be followed by investigation of the thermodynamic and structural properties as a function of pH of such expressed iso-2 mutants. If our model were proved by these studies, a way would be opened for a new understanding of the structure-function of proteins in which our polarizable hydrophobic core domain model would play a critical role. This would, in turn, help prediction of the structure-function of proteins based on the DNA sequences of their genes, an important goal in the biomedical science.